Histone deacetylase inhibitors reduce VEGF production and induce growth suppression and apoptosis in human mantle cell lymphoma

OBJECTIVES: Mantle cell lymphoma (MCL) is an incurable disease with an aggressive course and novel treatment strategies are urgently needed. The purpose of this study was to evaluate the effects of histone deacetylase (HDAC) inhibitors, a new group of antiproliferative agents, on human MCL cells. METHODS: Three MCL cell lines (JeKo-1, Hbl-2 and Granta-519) were exposed to different concentrations of the HDAC inhibitors sodium butyrate (NaB) and suberoylanilide hydroxamic acid (SAHA) for 8-72 h. Their effects on cell viability, apoptosis induction and cell cycle proliferation were studied. Moreover, the influence of SAHA on the expression of cyclin D1, the cell cycle regulators p21 and p27 and the production of vascular endothelial growth factor (VEGF) were analyzed. RESULTS: The HDAC inhibitors induced accumulation of acetylated histones in MCL cells. MTT assays and Annexin-V staining showed that they potently inhibited viability in a dose-dependent manner and induced apoptosis in all cell lines tested. Cell cycle analysis indicated that their exposure to SAHA or NaB decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases. Incubation with the two HDAC inhibitors resulted in downregulation of cyclin D1. SAHA lead to an upregulation of p21 in all cell lines and an upregulation of p27 in JeKo-1 and Granta-519 cells, while expression of p27 in Hbl-2 was not altered. In addition, SAHA inhibited the production of the angiogenic cytokine VEGF. Treatment with NaB increased the expression of p21 in JeKo-1 and Hbl-2 cells, while in Granta 519 cells no effect was noted. The expression of p27 remained constant in all three cell lines after exposure to NaB. CONCLUSION: Based on these findings, we provide evidence that HDAC inhibitors have antiproliferative effects in MCL and may represent a promising therapeutic approach.     

S100A4 regulates motility and invasiveness of human esophageal squamous cell carcinoma through modulating the AKT/Slug signal pathway

The involvement of S100A4 in modulating invasiveness of esophageal squamous cell carcinoma (ESCC) cell lines was explored. It was shown that S100A4 expression is positively correlated with the degree of invasiveness in human ESCC cells. The S100A4‐rich EC‐1 cells displayed higher migratory and invasive cell behavior while ET‐1 cells with low S100A4 expression levels displayed lower migratory and invasive cell behavior. S100A4 silencing by small interfering (siRNA) in EC‐1 cells induced E‐cadherin expression, and overexpression of S100A4 in a lowly invasive TE‐1 cells suppressed E‐cadherin expression. It is suggested that S100A4 silencing inhibit invasion via E‐cadherin upregulation, and overexpression of S100A4 promote invasion via E‐cadherin downregulation in ESCC cells. Compared with the vector‐transfected cells, S100A4 silencing in EC‐1 cells showed reduced ability of migration and invasiveness, and overexpression of S100A4 in TE‐1 cells showed increased ability of migration and invasiveness via wound‐healing and Transwell assay, and pseudometastatic model assay. Furthermore, re‐expression of S100A4 could increase the invasive phenotypes in S100A4 siRNA transfected EC‐1 cells, and S100A4 silencing could decrease the invasive phenotypes in S100A4 circular DNA (cDNA) transfected TE‐1 cells. It was found that Slug is downregulated in S100A4 siRNA transfected EC‐1 cells, and Slug is upregulated in S100A4 cDNA transfected TE‐1 cells. It was also discovered S100A4 cDNA induced protein kinase B (AKT) phosphorylation at Serine‐473(phospho‐AKT [p‐AKT]) levels, followed by the Slug upregulation, and S100A4 siRNA decreases the phospho‐AKT levels, followed by the Slug downregulation. The data suggested that S100A4 could regulate migratory and invasive behavior of human ESCC cells through modulating AKT/Slug pathway.     

Long noncoding RNA,tissue differentiation‐inducing nonprotein coding RNA is upregulated and promotes development of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the major causes of cancer death worldwide, especially in Eastern Asia. Due to the poor prognosis, it is necessary to further dissect the underlying mechanisms and explore therapeutic targets of ESCC. Recently, studies show that long noncoding RNAs (lncRNAs) have critical roles in diverse biological processes, including tumorigenesis. Increasing evidence indicates that some lncRNAs are widely involved in the development and progression of ESCC, such as HOTAIR, SPRY4‐IT1 and POU3F3. An emerging lncRNA, tissue differentiation‐inducing nonprotein coding RNA (TINCR), has been studied in human cutaneous squamous cell carcinoma and has critical biological function, but its role in ESCC remains unknown. Here, we evaluated the expression profile of TINCR and its biological function in ESCC. In a cohort of 56 patients, TINCR was significantly overexpressed in ESCC tissues compared with paired adjacent normal tissues. Further, in vitro silencing TINCR via small interfering RNA (siRNA) inhibited the proliferation, migration and invasion of ESCC cells. Meantime, siRNA treatment induced apoptosis and blocked the progression of cell cycle. Taken together, our study suggests that TINCR promotes proliferation, migration and invasion of ESCC cells, acting as a potential oncogene of ESCC.     

Clinicopathological significance of cyclooxygenase‐2 and cell cycle‐regulatory proteins expression in patients with esophageal squamous cell carcinoma

The aim of this study was to examine the expression of the molecular markers cyclooxygenase‐2 (COX‐2), Ki‐67, cyclin A, and p27 in patients with esophageal squamous cell carcinoma (ESCC), to ascertain the relationship of these makers with the clinicopathological significance of the patients, and to assess the additional prognostic value of the expression profile of these proteins for ESCC patients. The expression levels of COX‐2, Ki‐67, cyclin A, and p27 proteins of a series of primarily resected ESCC samples were determined by immunohistochemistry method. Clinicopathological and molecular factors affecting survival were analyzed by multivariate analysis. A total of 78 specimens were included in this study. Expression of COX‐2 was observed in 43 (55.1%) cases, and high levels of expression of Ki‐67, p27, and cyclin A were observed in 57 (73.0%), 33 (42.3%), 43 (55.1%) cases, respectively. The results of univariate survival analysis indicated that more advanced tumor stage, lymph node involvement, systemic dissemination, the levels of expression of COX‐2, Ki‐67, cyclin A, and p27 were associated with survival (all P‐value < 0.05). Multifactorial survival analysis revealed that only lymph node involvement, over‐expression of cyclin A, and low p27 expression were associated with the survival of the patients (hazard ratios = 2.83, 4.7, 2.9, respectively; P= 0.025, 0.042, 0.005, respectively). Among the molecular markers assessed, the expression of cell proliferation markers cyclin A and p27 are independent prognostic factors in patients with ESCC, whereas neither COX‐2 nor Ki‐67 is of independent prognostic value.     

Independent histological risk factors for lymph node metastasis of superficial esophageal squamous cell carcinoma; implication of claudin‐5 immunohistochemistry for expanding the indications of endoscopic resection

Endoscopic resection is curative for superficial esophageal squamous cell carcinoma (ESCC) limited to the lamina propria. Endoscopic resection is not recommended for superficial ESCC invading muscularis mucosa or submucosa, however, because of the high frequency of lymph node metastasis (LNM) in such patients. Methods to more accurately predict LNM by analysis of endoscopically resected specimens are needed. Patients with superficial ESCC who underwent surgery without prior chemoradiotherapy (n= 110) were retrospectively examined to determine whether LNM correlated with immunohistochemical parameters and conventional histological parameters, including depth of invasion and vascular permeation. Cancer cell expression of claudins‐1, 5, and 7, E‐cadherin, β‐catenin, and matrix metalloproteinase 7 was evaluated. Univariate analysis revealed that LNM correlated with claudin‐5 expression, but not any other immunohistochemical parameter examined. Multivariate analysis revealed three independent risk factors for LNM: aberrant claudin‐5 expression in cancer cells (odds ratio; OR [95% confidence interval]= 4.61[1.44–14.77]), depth of submucosal invasion greater than 200 µm (3.55 [1.02–13.17]), and positive lymphatic permeation (3.34 [1.22–9.15]). LNM was found in one of 29 (3.4%) patients with none of these three risk factors, and in 32 of 81 (39.5%) patients with one or more of these risk factors. In superficial ESCC, routine analysis of claudin‐5 expression in cancer cells together with depth of invasion and lymphatic permeation may be useful for predicting LNM and thereby reducing the number of patients undergoing additional surgery after successful endoscopic resection.     

Biglycan is overexpressed in pancreatic cancer and induces G1-arrest in pancreatic cancer cell lines

BACKGROUND & AIMS: Biglycan (PG-I), a component of the extracellular matrix (ECM), is overexpressed in pancreatic cancer. To determine possible matrix-tumor interactions, we investigated the effects of PG-I on pancreatic cancer. METHODS: PG-I expression in cell lines and tissue samples was examined by Northern blot and immunofluorescence. The effect of PG-I on proliferation was determined by measuring activity of Ras, ERK, Rb, [(3)H]-thymidine incorporation, and cell cycle analysis. Expression of cyclin A, B1, D1, E1, G1, PCNA, p21, and p27 was analyzed by Northern and Western blots. RESULTS: PG-I was overexpressed in the ECM of pancreatic cancer samples compared with normal pancreas or chronic pancreatitis tissues. Addition of transforming growth factor (TGF)-beta induced PG-I expression in HFL and HFFF2 fibroblasts as well as in the pancreatic cancer cell line PANC-1. PG-I inhibited growth of both TGF-beta-responsive and TGF-beta-unresponsive pancreatic cancer cells by inducing G1-arrest, which is accompanied by an increase of p27 and reduction of cyclin A and proliferating cell nuclear antigen. Furthermore, endogenous Ras and ERK activation was partly reduced by PG-I in vitro. CONCLUSIONS: The ECM protein PG-I inhibits growth by arresting pancreatic cancer cells in G1 and may be part of a host defense mechanism aimed at slowing down pancreatic tumor progression.     

KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97‐H cells in vitro and in animal models

Background: Downregulation of KAI1 gene expression has been found in many types of cancer cells and is closely related to cancer invasion and metastasis. This study was aimed at investigating the effects and possible underlying mechanisms of KAI1 gene on invasion and metastasis of human hepatocellular carcinoma (HCC). Methods: The invasive ability, visco‐elastic properties and cell adhesion forces were analysed in different HCC cells originating from the MHCC97‐H cell line transfected with either the sense or the antisense KAI1 expression plasmid. Tumuorigenicity, metastatic abilities, extracellular matrix (ECM) and intercellular adhesion molecule‐1 (ICAM‐1) expression were also evaluated in the nude mouse models of the xenografted and orthotopic liver cancer cells. Results: Compared with their parental cells, in the HCC cells transfected with the sense KAI1 gene, the invasive ability in vitro was significantly decreased (P<0.01); the cellular elastic coefficients K₁, K₂ and μ were significantly higher (P<0.05); the cells adhesion forces to fibronectin were significantly lower (P<0.01). The sense KAI1 gene transfection into the cancer cells also inhibited their invasion and lung metastasis in the orthotopic liver cancer nude mice. However, the opposite changes were observed in the HCC cells transfected with the antisense KAI1 gene. KAI1 gene transfection also affected ECM and ICAM‐1 expression in the transplanted liver cancer. Conclusion: The KAI1 gene plays an important role in the invasion and metastasis of human HCC and its upregulation in HCC cells suppresses their invasive and metastatic abilities. KAI1 gene functioned as a metastasis inhibitor by regulating the HCC cell biophysical behaviours including aggregation, adhesion, motility and visco‐elastic properties.     

Suberoylanilide hydroxamic acid (SAHA) and cladribine synergistically induce apoptosis in NK‐LGL leukaemia

Natural killer (NK) large granular lymphocyte (LGL) leukaemia features a clonal proliferation of CD3^? NK cells that can be classified into either aggressive or chronic categories. The NKL cell line, derived from an aggressive Asian NK cell leukaemia, and patient samples from chronic NK‐LGL leukaemia were used in our study to probe for synergistic efficacy of the epigenetic drugs vorinostat (SAHA) and cladribine in this disease. We demonstrate that histone deacetylases (HDACs) are over‐expressed in both aggressive and chronic NK leukaemia. Administration of the HDAC inhibitor SAHA reduces class I and II HDAC expression and enhances histone acetylation in leukaemic NK cells. In vitro combination treatment with SAHA and cladribine dose‐dependently exerts synergistic cytotoxic and apoptotic effects on leukaemic NK cells. Expression profiling of apoptotic regulatory genes suggests that both compounds led to caspase‐dependent apoptosis through activation of intrinsic mitochondrial and extrinsic death receptor pathways. Collectively, these data show that combined epigenetic therapy, using HDAC and DNA methyltransferase inhibitors, may be a promising therapeutic approach for NK‐LGL leukaemia.     

Molecular sequelae of histone deacetylase inhibition in human malignant B cells

Histone acetylation modulates gene expression, cellular differentiation, and survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells. In this study, we characterized the effect of suberoylanilide hydroxamic acid (SAHA), the prototype of a series of hydroxamic acid-based HDAC inhibitors, in cell lines and patient cells from B-cell malignancies, including multiple myeloma (MM) and related disorders. SAHA induced apoptosis in all tumor cells tested, with increased p21 and p53 protein levels and dephosphorylation of Rb. We also detected cleavage of Bid, suggesting a role for Bcl-2 family members in regulation of SAHA-induced cell death. Transfection of Bcl-2 cDNA into MM.1S cells completely abrogated SAHA-induced apoptosis, confirming its protective role. SAHA did not induce cleavage of caspase-8, -9, or -3 in MM.1S cells during the early phase of apoptosis, and the pan-caspase inhibitor ZVAD-FMK did not protect against SAHA. Conversely, poly(ADP)ribose polymerase (PARP) was cleaved in a pattern indicative of calpain activation, and the calpain inhibitor calpeptin abrogated SAHA-induced cell death. Importantly, SAHA sensitized MM.1S cells to death receptor-mediated apoptosis and inhibited the secretion of interleukin 6 (IL-6) induced in bone marrow stromal cells (BMSCs) by binding of MM cells, suggesting that it can overcome cell adhesion-mediated drug resistance. Our studies delineate the mechanisms whereby HDAC inhibitors mediate anti-MM activity and overcome drug resistance in the BM milieu and provide the framework for clinical evaluation of SAHA, which is bioavailable, well tolerated, and bioactive after oral administration, to improve patient outcome.     

Suberoylanilide hydroxamic acid (SAHA; vorinostat) suppresses translation of cyclin D1 in mantle cell lymphoma cells

Mantle cell lymphoma (MCL) has a chromosomal translocation resulting in the expression of the cyclin D1 gene driven by the powerful enhancer of the immunoglobulin heavy chain gene, leading to uncontrolled, overexpressed cyclin D1 protein. We showed that suberoylanilide hydroxamic acid (SAHA; vorinostat), one of the histone deacetylase inhibitors derived from hydroxamic acid, caused a dramatic decrease (90%) in protein levels of cyclin D1 after 8-hour exposure to SAHA (5 muM) in MCL lines (SP49, SP53, Jeko1). mRNA levels and protein stability of cyclin D1 were minimally affected by SAHA over 8 hours. In contrast, metabolic labeling assays showed that SAHA decreased incorporation of [(35)S]methionine into cyclin D1 protein. The drug also decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR), and eukaryotic translation initiation factor 4E binding protein (eIF4E-BP) and lowered the cap site binding activity of eIF4E in the MCL cells. In vitro phosphatidyl inositol (PI) kinase assay demonstrated that SAHA directly inhibited kinase activity of PI 3' kinase. Taken together, SAHA caused a rapid decrease of cyclin D1 in MCL by blocking the translation of cyclin D1 by inhibiting the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/eIF4E-BP pathway, probably by PI3K inhibition.     

The role of p27(Kip1) phosphorylation at serine 10 in the migration of malignant glioma cells in vitro

Until recently, Cip/Kip members were almost solely viewed as nuclear proteins with a principal function of inhibiting cyclin/cyclin dependent kinase (CDK) activity and hence, inhibiting cell cycle progression. P27(Kip1) (hereafter p27) belongs to the Cip/Kip family that binds and inhibits all the cyclin/CDK complexes, thus often referred as a universal CDK inhibitor. However, emerging studies now suggest that Cip/Kip proteins play additional roles outside of the nucleus. Indeed, previous reports have linked p27 to the regulation of actin dynamics and cell migration. In this study, we constructed a model of migration-activated glioma cells by using the migration-stimulating substrate, a kind of ECM, laminin in vitro. Our results present evidence that laminin drives glioma cell migration without altering cell proliferation. Further, actively migrating cells which expressioned high phosphorylation of p27 at Ser10, and induced its cytoplasmic localization. In this process, Jab1 and CRM1 were also involved. Thus phosphorylation of p27 at Ser10 is necessary for both cytoplasmic localization and induction of cell migration. These observations solidified a genetic role of p27 in cell migration and this was independent of cyclin/CDK inhibition. Eventually, we transiently transfected p27S10A into T98G glioma cells, found that overexpression of p27S10A inhibited cell migration but not cell proliferation. These data linked phosphorylation of p27 at Ser10 and cell motility. Therefore, the major phosphorylation site at Ser10 of p27 played a pivotal role in the migration of malignant glioma cells.     

The novel inhibitor of histone deacetylase resminostat (RAS2410) inhibits proliferation and induces apoptosis in multiple myeloma (MM) cells

Inhibition of histone deacetylase (HDAC) is a promising mechanism for novel, anti‐myeloma agents. We investigated the effects of the novel HDAC inhibitor resminostat on multiple myeloma (MM) cells in vitro. Resminostat is a potent inhibitor of HDACs 1, 3 and 6 [50% inhibitory concentration (IC₅₀) = 43–72 nmol/l] representing HDAC classes I and II and induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of resminostat abrogated cell growth and strongly induced apoptosis (IC₅₀ = 2·5–3 μmol/l in 3 out of 4 MM cell lines) in MM cell lines as well as primary MM cells. At 1 μmol/l, resminostat inhibited proliferation and induced G0/G1 cell cycle arrest in 3 out of 4 MM cell lines accompanied with decreased levels of cyclin D1, cdc25a, Cdk4 and pRb as well as upregulation of p21. Resminostat decreased phosphorylation of 4E‐BP1 and p70S6k indicating an interference with Akt pathway signalling. Treatment with resminostat resulted in increased protein levels of Bim and Bax and decreased levels of Bcl‐xL. Caspases 3, 8 and 9 were activated by resminostat. Furthermore, synergistic effects were observed for combinations of resminostat with melphalan and the proteasome inhibitors bortezomib and S‐2209. In conclusion, we have identified potent anti‐myeloma activity for this novel HDAC inhibitor.     

HDAC1对乳腺癌细胞MDA—MB-435s增殖影响的研究

目的研究组蛋白去乙酰化酶1（HDAC1）对乳腺癌细胞株MDA—MB-435s增殖周期的影响。方法培养雌激素受体阴性乳腺癌细胞株MDA—MB-435s,转染HDAC1作为正向干预,加入组蛋白去乙酰化酶抑制剂SAHA负向干预,倒置相差显微镜观察细胞形态改变,MTT比色法观察细胞生长情况,流式细胞术检测细胞周期变化。结果转染HDAC1质粒后,乳腺癌细胞株S期细胞比例显著增加,而加入SAHA后细胞增殖受到抑制,细胞阻滞在G0／G1期。结论HDAC1可促进乳腺癌细胞的增殖,组蛋白去乙酰化酶抑制剂能阻止其增殖。     

Combination of SAHA and bortezomib up‐regulates CDKN2A and CDKN1A and induces apoptosis of Epstein‐Barr virus‐positive Wp‐restricted Burkitt lymphoma and lymphoblastoid cell lines

Epstein‐Barr virus (EBV) latent proteins exert anti‐apoptotic effects on EBV‐transformed lymphoid cells by down‐regulating BCL2L11 (BIM), CDKN2A (p16^INK4A) and CDKN1A (p21^WAF1). However, the potential therapeutic effects of targeting these anti‐apoptotic mechanisms remain unexplored. Here, we tested both in vitro and in vivo effects of the combination of histone deacetylase (HDAC) and proteasome inhibitors on the apoptosis of six endemic Burkitt lymphoma (BL) lines of different latency patterns (types I and III and Wp‐restricted) and three lymphoblastoid cell lines (LCLs). We found that the combination of HDAC and proteasome inhibitors (e.g. SAHA/bortezomib) synergistically induced the killing of Wp‐restricted and latency III BL and LCLs but not latency I BL cells. The synergistic killing was due to apoptosis, as evidenced by the high percentage of annexin V positivity and strong cleavage of PARP1 (PARP) and CASP3 (caspase‐3). Concomitantly, SAHA/bortezomib up‐regulated the expression of CDKN2A and CDKN1A but did not affect the level of BCL2L11 or BHRF1 (viral homologue of BCL2). The apoptotic effects were dependent on reactive oxygen species generation. Furthermore, SAHA/bortezomib suppressed the growth of Wp‐restricted BL xenografts in nude mice. This study provides the rationale to test the novel application of SAHA/bortezomib on the treatment of EBV‐associated Wp‐restricted BL and post‐transplant lymphoproliferative disorder.     

Xiaotan Sanjie decoction inhibits interleukin-8-induced metastatic potency in gastric cancer

AIM:To investigate the interaction between Xiaotan Sanjie(XTSJ) decoction and interleukin-8(IL-8) and its effect on adhesion,migration and invasion of SGC-7901 gastric cancer cells.METHODS:SGC-7901 gastric cancer cells were exposed to serum containing XTSJ decoction and/orIL-8(1 ng/m L).SGC-7901 cell adhesion to fibronectin,an extracellular matrix component,was detected using the Cell Counting Kit-8.Migration and invasion abilities of SGC-7901 cells were detected by scratch wound and Transwell chamber assays.Then,protein(immunofluorescence and Western blot) and m RNA levels(quantitative polymerase chain reaction) of cluster of differentiation 44(CD44),a cell adhesion molecule,were measured in 72-h-cultured SGC-7901 cells.RESULTS:Cell adhesion was promoted by IL-8(P = 0.001),but was inhibited by XTSJ decoction(P = 0.0001).Similarly,IL-8 promoted SGC-7901 cell invasion(P = 0.003),and XTSJ decoction inhibited cell invasion(P = 0.001).IL-8 induced SGC-7901 cell migration,but this was inhibited by XTSJ decoction.IL-8 up-regulated CD44 protein(P = 0.028) and m RNA expression(P = 0.002),whereas XTSJ decoction inhibited CD44 protein expression(P = 0.0001),but not m RNA expression(P = 0.275).An interaction between XTSJ decoction and IL-8 was confirmed in the invasion(P = 0.001) and CD44 m RNA expression of SGC-7901 cells(P = 0.010),but not in cell adhesion(P = 0.051).CONCLUSION:XTSJ decoction may inhibit adhesion,migration and invasion of gastric cancer cells,which is partly associated with down-regulation of IL-8.